Microscopy with light, electrons, and nanomechanical probes has revolutionized biology, yet at room temperature, the potential of many techniques can not be fully exploited due to thermal noise, radiation damage, or mechanical interactions with the object. Cryogenic imaging has advanced greatly in recent years, but the fixation of biological specimens for this purpose is a difficult challenge due to the need to prevent ice crystal formation upon cooling.
A conventional method of vitrifying hydrated samples (i.e. freezing while avoiding ice crystallization) is high pressure freezing, which is a laborious procedure that needs to be executed by a highly skilled operator to yield reproducible results (see J. Dubochet “The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells” in “Methods in Cell Biology, vol. 79: Cellular Electron Microscopy” (J. R. McIntosh), Academic Press, 2007). For biological samples, damage may also be induced by the application of high pressure (A. Leforestier et al. “Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure” in “Journal of Microscopy”, vol. 184, 1996, p. 4-13) (˜2000 bar is typical), and cells need to be removed from the environment in which they are cultured and often need to be embedded in a medium other than water for the freezing operation. Subtle alterations of cell state due to these manipulations are inherently unavoidable, and have therefore always been tacitly accepted by the community. In-situ ultra-rapid freezing eliminates disturbances (other than the rapid cooling itself) altogether, and is therefore expected to reveal a wealth of new biological insights that would otherwise remain obscured by the artifacts of sample preparation.
At atmospheric pressure, vitrification can be achieved by ultra-rapid freezing if the cooling rate is sufficiently high; the critical rate required depends on the concentration of natural or synthetic cryoprotective agents, such as, for example, salt, proteins, Dimethyl sulfoxide, or 1,2-propanediol. To prevent ice crystallization, water needs to be vitrified at cooling rates of ˜106 K/s if no cryoprotective agent is added (R. Risco et al. “Thermal performance of qurtz capillaries for vitrification” in “Cryobiology” vol. 55, 2007, p. 222-229). However such rapid cooling currently is far beyond the limitations of conventional techniques; therefore, the requirement is usually relaxed by adding cryo-protectants, which may be toxic to the cell and can subtly alter physical structure and biochemical composition. The attractiveness of rapid cooling is then significantly diminished, since the process is not guaranteed to yield a faithful representation of the object in its natural state. Ultra-rapid cooling removes this limitation.
Large cooling rates have been obtained with a method of ultra-rapid freezing for cell cryopreservation as described in U.S. Pat. No. 6,300,130 B1 (and U.S. Pat. No. 6,403,376 B1). Biological material is placed in thermal contact with a cryogenically coolable environment, while radiation energy is applied to the biological material for melting a portion thereof. Rapid interruption of the irradiation results in a rapid cooling and vitrification of the biological material. This freezing technique has the following disadvantages. Firstly, the biological material is directly heated by the irradiation so that a radiation source has to be adapted to the absorption of the biological material resulting in a restricted application of a freezing equipment. Furthermore, damages of the biological material may occur. Thus, only a portion of cells included in the biological material may survive (about 80%). Further restrictions result from the conventional heating with a radiation beam focussed into the sample. Point-shaped vitrified sample regions can be obtained only. Also, when cells are thawed by the focused radiation source, the surrounding medium will still be frozen, preventing facile access to the sample for the purpose of exchanging media or experimental manipulations. Furthermore, a freezing front is created, which moves from a cool boundary of this region to a center thereof after switching-off the irradiation. An inhomogeneous sample structure may result even in the center of the vitrified region. Similarly, the inhomogeneous temperature distribution in the thawed state is problematic for live samples, which will be exposed to temperature gradients from the freezing point of water to approximately 40° C. over distances of the order of 10 μm.
Efforts for obtaining efficient cooling with large cooling rates have been made not only in the field of ultra-rapid freezing of biological samples but also in other technologies, like electronics, sensor techniques and chemical engineering, e.g. for switching operation conditions or adjusting reaction conditions of a chemical reaction.